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Establishing an ANO1-Based Cell Model for High-Throughput Screening Targeting TRPV4 Regulators
Kai Zheng1, Jiang Hu1, Cheng Hu1
1College of Laboratory Medicine, Jilin Medical University, Jilin 132000, China.
Abstract:
Transient receptor potential vanilloid 4 (TRPV4) is a widely expressed cation channel that plays an important role in many physiological and pathological processes. However, most TRPV4 drugs carry a risk of side effects. Moreover, existing screening methods are not suitable for the high-throughput screening (HTS) of drugs. In this study, a cell model and HTS method for targeting TRPV4 channel drugs were established based on a calcium-activated chloride channel protein 1 Anoctamin 1 (ANO1) and a double mutant (YFP-H148Q/I152L) of the yellow fluorescent protein (YFP). Patch-clamp experiments and fluorescence quenching kinetic experiments were used to verify that the model could sensitively detect changes in intracellular Ca2+ concentration. The functionality of the TRPV4 cell model was examined through temperature variations and different concentrations of TRPV4 modulators, and the performance of the model in HTS was also evaluated. The model was able to sensitively detect changes in the intracellular Ca2+ concentration and also excelled at screening TRPV4 drugs, and the model was more suitable for HTS. We successfully constructed a drug cell screening model targeting the TRPV4 channel, which provides a tool to study the pathophysiological functions of TRPV4 in vitro.
Insights
Researchers developed a new cell model for high-throughput screening (HTS) of Transient Receptor Potential Vanilloid 4 (TRPV4) drugs. This innovative method efficiently identifies potential TRPV4 therapeutics with reduced side effects, advancing drug discovery.
Area of Science:
- Pharmacology
- Molecular Biology
- Biophysics
Background:
- Transient Receptor Potential Vanilloid 4 (TRPV4) channels are crucial in physiological and pathological processes.
- Current TRPV4 drug development faces challenges due to side effects and limitations in high-throughput screening (HTS) methods.
Purpose of the Study:
- To establish a novel cell model and HTS method for identifying drugs targeting the TRPV4 channel.
- To overcome the limitations of existing screening techniques and facilitate efficient drug discovery for TRPV4-related conditions.
Main Methods:
- Development of a cell model utilizing calcium-activated chloride channel protein 1 (ANO1) and a yellow fluorescent protein (YFP) double mutant.
- Validation using patch-clamp and fluorescence quenching kinetic experiments to assess intracellular Ca2+ concentration changes.
- Evaluation of the model's functionality with temperature variations and TRPV4 modulators for HTS performance.
Main Results:
- The developed cell model demonstrated high sensitivity in detecting intracellular Ca2+ concentration fluctuations.
- The model proved effective for HTS, successfully screening TRPV4 drugs.
- The established system is well-suited for large-scale screening of potential TRPV4 therapeutics.
Conclusions:
- A robust cell-based drug screening model for the TRPV4 channel has been successfully constructed.
- This model offers a valuable tool for in vitro studies of TRPV4's pathophysiological roles.
- The developed HTS method enhances the efficiency and applicability of TRPV4 drug discovery.
